Micro4 microsyringe controller

Mice received a 2 μl ivt injection of BDNF (Peprotech Laboratories, UK), diluted at 1 mg/ml in 0.01 M phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA, PanReac Applichem, Germany) (BSA-PBS). Ivt injections were performed using a glass capillary with a 50–70 μm outer diameter, connected to a Hamilton syringe and a Micro4 Microsyringe controller (World Precision Instruments, FL, USA), at a rate of 500 nl/s. Control groups received an ivt injection of 2 μl vehicle (BSA-PBS). Two main treatment paradigms were used: (i) a single dose immediately after crush with sacrifice at 7 or 14 days post-injury (dpi), and (ii) repeated ivt injection at 0, 3, and 5 d with sacrifice at 7 dpi, or at 0, 5, and 10 d with sacrifice at 14 d post ONC.

Intraorbital ONC was induced in the left experimental eye, while the right eye served as an internal control, using previously established methods [24 (link),25 (link),57 (link)]. Briefly, the optic nerve was accessed by an incision in the conjunctiva temporally around the eye. The left optic nerve was crushed at 1 mm from the optic disk for 0.5 s, applying a constant and consistent force using cross-action forceps. Before and after the ONC procedure, funduscopy was performed to ensure a normal retinal perfusion. Ivt injection of 1 µL of the MMP-12 inhibitor at concentrations of 2 mM (10% DMSO in PBS), were performed at the day of surgery and 2 days post ONC (dpi) in the treated group. The vehicle control group received ivt injections of 1 μL vehicle only (10% DMSO in PBS) at similar time points. Ivt injections were performed using a glass capillary with a 50–70 μm outer diameter, connected to a Micro4 Microsyringe controller (World Precision Instruments, Freidberg, Germany), at a rate of 500 nL/s. Animals were sacrificed at 4 dpi.